Prefixation for Increased Rare Cell Recovery

ABSTRACT

Traditionally, enriching or isolating rare circulating cells from a sample has proven difficult as rare circulating cells can be heterogeneous, which limits the use of techniques based on biological properties (e.g., cell surface markers), and can be flexible, which hinders techniques based on physical properties (e.g., diameter size). Methods disclosed herein are capable of enriching or isolating rare circulating cells are minimally impacted by heterogeneity or flexibility of rare circulating cells, and therefore, overcome disadvantages observed with prior techniques. For example, a method for enriching or isolating rare circulating cells from a sample of a subject including fixing one or more rare circulating cells in a sample to create a fixed sample, and isolating the one or more rare circulating cell from the fixed sample using a size exclusion technique is disclosed.

BACKGROUND

Cancer can start in any organ or tissue in the body. A primary cancer ortumor is a first, original tumor that develops in the body. A metastaticcancer or tumor occurs when cancer cells spread from a primary tumor toa new part of the body. In other words, metastatic tumors always startfrom cancer cells in another part of the body. For example, breastcancer can spread from its primary site (i.e., a breast) to form a newtumor in a different part of the body, such as bones. Cancer cells in asecond bone tumor are the same as cells in the primary breasttumor—i.e., cells are breast cancer cells, and not bone cancer cells.

Cancer spreads from a primary tumor to other parts of the body viacirculating tumor cells (CTCs). CTCs arise from primary or secondarytumors and are shed into the vasculature system from the tumors. CTCstravel in blood or lymph fluid to distant sites to form metastasis aftergaining properties of extravasation, survival and proliferation in atarget tissue.

SUMMARY

Compositions and methods disclosed herein encompass the discovery thatfixing rare circulating cells in a sample from a subject increases arecovery rate of rare circulating cells from a sample. Rare circulatingcells can be recovered from a sample using, for example, size exclusiontechniques (e.g., filtration). However, cells are elastic, and a cellcan pass through a pore having a diameter smaller than a diameter of acell, which, in turn, can compromise recovery. Without being bound toany theory, fixation of a rare circulating cell may increase therigidity of the cell, and therefore, make it less likely the fixed rarecirculating cell will be able to pass through a pore having a diametersmaller than a diameter of the cell, which can increase recovery.

In one aspect, a method for isolating or enriching rare circulatingcells from a sample can include fixing one or more rare circulatingcells in a sample. In some embodiments, rare circulating cells caninclude circulating tumor cells, epithelial progenitor cells, stemcells, mesenchymal cells, fetal cells, or combinations of these celltypes. In some embodiments, rare circulating cells can includecirculating tumor cells. In some embodiments, circulating tumor cellscan originate from a carcinoma, sarcoma, leukemia, lymphoma, myeloma ornervous system cancer. In a preferred embodiment, circulating tumorcells can originate from a carcinoma, for example, a basal cellcarcinoma, a squamous cell carcinoma, a renal cell carcinoma, aninvasive ductal carcinoma or an adenocarcinoma.

In some embodiments, a sample can be a blood sample, for example, awhole blood sample, a plasma sample or a serum sample. In someembodiments, a sample can be a lymph fluid sample. In some embodiments,a sample can be from a subject, for example, a mammal, preferably, ahuman.

In some embodiments, fixing one or more rare circulating cells in asample can include adding a fixative to a sample. In some embodiments, afixative can include an aldehyde, for example, formaldehyde,glutaraldehyde, or paraformaldehyde. In a preferred embodiment, afixative can include formaldehyde.

In some embodiments, fixing one or more rare circulating cells in asample can include adding a fixative to the sample so that aconcentration of the fixative in the sample can be at least 0.05% w/v,at least 0.10% w/v, at least 0.15% w/v, at least 0.20% w/v, or at least0.25% w/v. In some embodiments, fixing one or more rare circulatingcells in a sample can include adding a fixative to the sample so that aconcentration of the fixative in the sample can be at most 0.40% w/v, atmost 0.35% w/v, at most 0.30% w/v, at most 0.25% w/v, or at most 0.20%w/v. In some embodiments, fixing one or more rare circulating cells in asample can include adding a fixative to the sample so that aconcentration of the fixative in the sample can be 0.05-0.35% w/v,preferably, 0.1-0.3% w/v, more preferably, about 0.25% w/v.

In some embodiments, fixing one or more rare circulating cells in asample can include adding at least 50 μL, at least 100 μL, at least 150μL, at least 200 μL, at least 250 μL, at least 500 μL or at least 1 mLof a fixative to the sample. In some embodiments, fixing one or morerare circulating cells in a sample can include adding at most 2 mL, atmost 1.5 mL, at most 1 mL, at most 500 μL, at most 250 μL, or at most100 μL of a fixative to the sample. In some embodiments, fixing one ormore rare circulating cells in a sample can include adding 50-500 μL,preferably, 20-500 μL, of a fixative to the sample.

In some embodiments, fixing one or more rare circulating cells in asample can include incubating the sample with a fixative. In someembodiments, a sample can be incubated with a fixative for at least 30seconds, at least 1 minute, at least 2 minutes, at least 5 minutes, orat least 10 minutes. In some embodiments, a sample can be incubated witha fixative for at most 30 minutes, at most 25 minutes, at most 20minutes, at most 15 minutes, or at most 10 minutes. In some embodiments,a sample can be incubated with a fixative for 1-20 minutes, preferably,3-10 minutes.

In some embodiments, fixing the one or more rare circulating cells cancreate a fixed sample (i.e., a sample that includes one or more fixedrare circulating cells).

In some embodiments, a method for isolating rare circulating cells froma sample can comprise isolating one or more rare circulating cells froma sample using a size exclusion technique. In some embodiments, a methodfor enriching rare circulating cells from a sample can compriseenriching rare circulating cells in a sample using a size exclusiontechnique. For example, one or more rare circulating cells can beisolated or enriched by directing a fixed sample through one or morefilter membranes. In some embodiments, one or more filter membranes canbe part of a microfiltration device. In some embodiments, at least oneof the one or more filter membranes can have a pore size of at least 5microns, at least 6 microns, at least 7 microns, at least 8 microns, atleast 9 microns, or at least 10 microns. In some embodiments, at leastone of the one or more filter membranes can have a pore size of at most12 microns, at most 11 microns, at most 10 microns, at most 9 microns,at most 8 microns, at most 7 microns, or at most 6 microns. In someembodiments, at least one of the one or more filter membranes can have apore size of between 5 and 10 microns, preferably, between 7 and 9microns, more preferably 8 microns.

In one aspect, a method of analyzing rare circulating cells in a sampleinclude fixing one or more rare circulating cells in a sample. In someembodiments, rare circulating cells can include circulating tumor cells,epithelial progenitor cells, stem cells, mesenchymal cells, fetal cells,or combinations of these cell types. In some embodiments, rarecirculating cells can include circulating tumor cells. In someembodiments, circulating tumor cells can originate from a carcinoma,sarcoma, leukemia, lymphoma, myeloma or nervous system cancer. In apreferred embodiment, circulating tumor cells can originate from acarcinoma, for example, a basal cell carcinoma, a squamous cellcarcinoma, a renal cell carcinoma, an invasive ductal carcinoma or anadenocarcinoma.

In some embodiments, a sample can be a blood sample, for example, awhole blood sample, a plasma sample or a serum sample. In someembodiments, a sample can be a lymph fluid sample. In some embodiments,a sample can be from a subject, for example, a mammal, preferably, ahuman.

In some embodiments, fixing one or more rare circulating cells in asample can include adding a fixative to the sample. In some embodiments,a fixative can include an aldehyde, for example, formaldehyde,glutaraldehyde, or paraformaldehyde. In a preferred embodiment, afixative can include formaldehyde.

In some embodiments, fixing one or more rare circulating cells in asample can include adding a fixative to the sample so that aconcentration of the fixative in the sample can be at least 0.05% w/v,at least 0.10% w/v, at least 0.15% w/v, at least 0.20% w/v, or at least0.25% w/v. In some embodiments, fixing one or more rare circulatingcells in a sample can include adding a fixative to the sample so that aconcentration of the fixative in the sample can be at most 0.40% w/v, atmost 0.35% w/v, at most 0.30% w/v, at most 0.25% w/v, or at most 0.20%w/v. In some embodiments, fixing one or more rare circulating cells in asample can include adding a fixative to the sample so that aconcentration of the fixative in the sample can be 0.05-0.35% w/v,preferably, 0.1-0.3% w/v, more preferably, about 0.25% w/v.

In some embodiments, fixing one or more rare circulating cells in asample can include adding at least 50 μL, at least 100 μL, at least 150μL, at least 200 μL, at least 250 μL, at least 500 μL or at least 1 mLof a fixative to the sample. In some embodiments, fixing one or morerare circulating cells in a sample can include adding at most 2 mL, atmost 1.5 mL, at most 1 mL, at most 500 μL, at most 250 μL, or at most100 μL of a fixative to the sample. In some embodiments, fixing one ormore rare circulating cells in a sample can include adding 50-500 μL,preferably, 20-500 μL, of a fixative to the sample.

In some embodiments, fixing one or more rare circulating cells in asample can include incubating the sample with a fixative. In someembodiments, a sample can be incubated with a fixative for at least 30seconds, at least 1 minute, at least 2 minutes, at least 5 minutes, orat least 10 minutes. In some embodiments, a sample can be incubated witha fixative for at most 30 minutes, at most 25 minutes, at most 20minutes, at most 15 minutes, or at most 10 minutes. In some embodiments,a sample can be incubated with a fixative for 1-20 minutes, preferably,3-10 minutes.

In some embodiments, fixing the one or more rare circulating cells cancreate a fixed sample.

In some embodiments, a method of analyzing rare circulating cells in asample can comprise enriching or isolating one or more rare circulatingcells from a sample. In some embodiments, one or more rare circulatingcells can be isolated or enriched using a size exclusion technique. Forexample, one or more rare circulating cells can be isolated or enrichedby directing a fixed sample through one or more filter membranes. Insome embodiments, one or more filter membranes can be part of amicrofiltration device. In some embodiments, at least one of the one ormore filter membranes can have a pore size of at least 5 microns, atleast 6 microns, at least 7 microns, at least 8 microns, at least 9microns, or at least 10 microns. In some embodiments, at least one ofthe one or more filter membranes can have a pore size of at most 12microns, at most 11 microns, at most 10 microns, at most 9 microns, atmost 8 microns, at most 7 microns, or at most 6 microns. In someembodiments, at least one of the one or more filter membranes can have apore size of between 5 and 10 microns, preferably, between 7 and 9microns, more preferably 8 microns.

In some embodiments, a method of analyzing rare circulating cells in asample can include performing an analysis of one or more rarecirculating cells. In some embodiments, performing an analysis of one ormore rare circulating cells can include staining, dyeing,immunocytochemistry, immunohistochemistry, in situ hybridization, PCR,single cell sequence analysis, cell sorting, cell counting, microscopyor a combination these techniques.

In one aspect, a composition can include one or more rare circulatingcells and a fixative.

In some embodiments, rare circulating cells can include circulatingtumor cells, epithelial progenitor cells, stem cells, mesenchymal cells,fetal cells, or combinations of these cell types. In some embodiments,rare circulating cells can include circulating tumor cells. In someembodiments, circulating tumor cells can originate from a carcinoma,sarcoma, leukemia, lymphoma, myeloma or nervous system cancer. In apreferred embodiment, circulating tumor cells can originate from acarcinoma, for example, a basal cell carcinoma, a squamous cellcarcinoma, a renal cell carcinoma, an invasive ductal carcinoma or anadenocarcinoma.

In some embodiments, a sample can be a blood sample, for example, awhole blood sample, a plasma sample or a serum sample. In someembodiments, a sample can be a lymph fluid sample. In some embodiments,a sample can be from a subject, for example, a mammal, preferably, ahuman.

In some embodiments, a fixative can include an aldehyde, for example,formaldehyde, glutaraldehyde, or paraformaldehyde. In a preferredembodiment, a fixative can include formaldehyde.

In some embodiments, a concentration of fixative in a composition can beat least 0.05% w/v, at least 0.10% w/v, at least 0.15% w/v, at least0.20% w/v, or at least 0.25% w/v. In some embodiments, a concentrationof fixative in a composition can be at most 0.40% w/v, at most 0.35%w/v, at most 0.30% w/v, at most 0.25% w/v, or at most 0.20% w/v. In someembodiments, a concentration of fixative in a composition can be0.05-0.35% w/v, preferably, 0.1-0.3% w/v, more preferably, about 0.25%w/v.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing a recovery of NCI-H226 cells with 0.2%formaldehyde pretreatment. Freshly trypsinized H226 cells were spikedinto whole blood collected with customized Transfix® tubes and includinga formaldehyde pretreatment. After performing a circulating tumor cell(CTC) isolation and detection process, NCI-H226 cells retained on afiltration membrane were counted, which is reflected on the graph.

FIG. 2 is a bar chart showing a recovery of NCI-H226 cells withindicated concentrations of formaldehyde pretreatment. Mean values with95% confidence bars are shown for conditions that were successfullytested in multiple replicates. No error bars are shown for single datapoints.

DEFINITIONS

About: As used herein, the term “approximately” or “about,” as appliedto one or more values of interest, refers to a value that is similar toa stated reference value. In certain embodiments, the term“approximately” or “about” refers to a range of values that fall within25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than orless than) of the stated reference value unless otherwise stated orotherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Enrich: As used herein, “enrich” or “enriching” refers to a process ofincreasing the concentration or proportion of rare circulating cells ina sample. In other words, a concentration or proportion of rarecirculating cells in a sample can be greater at a second time point thana concentration or proportion of rare circulating cells in a sample at afirst time point. For instance, “enriching” can include removing one ormore components from a sample, e.g., reducing the volume of a samplewithout a concomitant reduction in the amount of rare circulating cells,which can be achieved, e.g., by size exclusion techniques such asfiltration.

Isolate: As used herein, “isolate” or “isolating” refers to a process ofseparating rare circulating cells from at least one other component in asample. Isolated rare circulating cells may be separated from about 40%,about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,about 99%, or more than about 99% of other components in a sample. Insome embodiments, isolated rare circulating cells can be about 80%,about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about95%, about 96%, bout 97%, about 98%, about 99%, or more than about 99%pure. As used herein, a substance is “pure” if it is substantially freeof other components.

Rare circulating cells: As used herein, “rare circulating cells” referto cells that can be found with a frequency on the order of 100 cells orless per mL of a sample of a subject, for example, a blood or lymphfluid sample. Examples of rare circulating cells can include circulatingtumor cells, circulating epithelial progenitor cells, circulating stemcells, circulating mesenchymal cells, and circulating fetal cells. Morespecifically, examples of rare circulating cells of a solid tumor caninclude circulating tumor cells, cancer stem cells, and cells that aremigrating to a tumor (e.g., due to chemoattraction) such as circulatingendothelial progenitor cells, circulating endothelial cells, circulatingpro-angiogenic myeloid cells, and circulating dendritic cells.Circulating tumor cells, for instance, can be generally found with afrequency of 1-10 circulating tumor cells per mL of blood. Similarly,circulating fetal cells can be typically found with a frequency of 1-10circulating fetal cells per mL of blood.

DETAILED DESCRIPTION

It has been known that rare circulating cells can be found in biologicalfluids, such a blood and lymph fluid. For example, it has been known forover a century that circulating tumor cells (CTCs) are shed into thevasculature system from a primary tumor and circulate in bloodstreams todistant sites to form metastasis. Rare circulating cells in biologicalfluids can be obtained from in a non-invasive way (e.g., by a blooddraw). In the case of CTCs, isolating these cells from a biologicalfluid and analyzing CTCs can provide significant amount of informationfor a better understanding, e.g., of tumor biology. In this way, CTCs ina blood sample can be used as a “liquid biopsy” to follow patients overtime. In the case of circulating fetal cells, isolating these cells froma mother's blood and analyzing them can provide important geneticinformation about a developing fetus, for example, the presence ofaneuploidy. However, detection, enumeration and molecularcharacterization of rare circulating cells has been challenging as rarecirculating cells are, as their name suggests, rare. For example, thefrequencies of CTCs found in peripheral blood of metastatic patients hasbeen on the order of 1-10 CTC per mL. In comparison, 1 mL of bloodcontains millions of white blood cells and billions of red blood cells.For this reason, isolation or enrichment of rare circulating cells frombiological fluids can be important for examining them.

Generally, rare circulating cells isolation and enrichment methods canbe categorized to two classes:

1) Isolation or Enrichment Strategies Based on Biological Properties

Examples of biological properties used in isolation or enrichmentmethods can include cell surface biomarkers. This type of enrichmentstrategy is employed in the only FDA approved CTC in vitro diagnosticsystem, CellSearch™. While this type of strategy has seen some success,rare circulating cells, including CTCs, can be highly heterogeneous anddo not necessarily express, e.g., the same biomarker. As such,techniques based on biological properties are limited to enriching orisolating cells that include a preselected biological property, andtherefore, necessarily fail to retain cells that lack the preselectedbiological property. Consequently, rare circulating cells withunexpected or unique biological properties, e.g., may not be enriched orisolated using these techniques.

2) Isolation or Enrichment Strategies Based on the Physical Properties

Examples of isolation or enrichment strategies based on physicalproperties include, but not limited to, density gradients and sizeexclusion techniques, e.g., filtration through a pore having aparticular size. For example, Siemen's automated CTC platform(Integrated Cell Capture Micro-Device or ICCMD) utilizes filtration andsize to capture CTCs. An ICCMD platform can filter milliliters of bloodthrough one or more 8 um filter membranes and can retain CTCs on the oneor more filters for downstream analysis. This method relies on thelarger size of CTCs (e.g., around 30 μM in the case of breast cancercells) compared to the majority of blood cells, which can range from 8to 12 μM. However, cells are elastic, and cells (including rarecirculating cells) can still pass through a pore having a diametersmaller than that of the cell. For instance, Coumans, et al., reportedthat a 15 μm cell can easily pass through a 5 μm pore while a 6 μm rigidbead does not pass through a 5 μm pore. (Coumans, et al., “Filtrationparameters influencing circulating tumor cell enrichment from wholeblood,” PLoS One, 8(4), Apr. 26, 2013, which is incorporated byreference in its entirety). Rare circulating cells that pass through asmaller diameter may not retained, and if that occurs, recovery of rarecirculating cells can be compromised.

As current techniques for enriching or isolating rare circulating cellsfrom a sample based on biological properties (e.g., cell surfacemarkers) are limited by the heterogeneity of biological properties ofrare circulating cells, and current techniques for enriching orisolating rare circulating cells from a sample based on physicalproperties (e.g., size) are hindered by the flexibility of rarecirculating cells, there is a need for a method that can be appliedregardless of the biological properties of the rare circulating cellsand can attenuate the impact of cell flexibility on recovery rates.

Without being bound to any particular theory, methods and compositionsdescribed herein can fulfill a need in the art, as the methods andcompositions increase the rigidity of rare circulating cells byfixation, which permits the use of techniques for the separation of rarecirculating cell based on physical properties (e.g., size) with a highretention rate.

Samples Including Rare Circulating Cells

Rare circulating cells are cells that are found in biological fluids inextremely low numbers (i.e., 100 cells or less per mL of fluid sample).Typically, rare circulating cells are present in these low numbersbecause they are not normally found in the particular biological fluid,but rather, are present in the fluid as a result of an abnormalcondition (e.g., cancer, disease, pregnancy, etc.). Examples of rarecirculating cells can include circulating tumor cells, circulatingepithelial progenitor cells, circulating stem cells, circulatingmesenchymal cells, and circulating fetal cells.

Rare circulating cells can be present in a sample, most often abiological fluid that originated from a subject. A subject can be ahuman, but can be include another mammal such as, e.g., another primate,a rodent, a canine, a feline, an equine, an ovine, or a porcine. Asample can include, without limitation, whole blood, plasma, serum, redblood cells, white blood cells (e.g., peripheral blood mononuclearcells), cord blood, saliva, urine, stool (i.e., feces), sputum,bronchial lavage fluid, tears, nipple aspirate, lymph fluid, bonemarrow, amniotic fluid, spinal and pleural effusion, any other bodilyfluid, and cellular extracts thereof. In some embodiments, a sample canbe whole blood or a fractional component thereof, such as plasma, serum,or a cell pellet.

A sample can be obtained by any technique known in the art. For example,lymph fluid can be collected using a needle to draw fluid from a lymphnode or during a lymph node biopsy. As another example, a blood samplecan be collected by an intravenous blood draw.

Historically, a significant proportion of biological samples have beencollected in one place and transported to another place for analysis.Accordingly, once a sample has been obtained, the sample may need to betransported and/or stored. Consequently, a sample may be added to astorage solution at the time the sample is obtained from a subject. Forexample, when blood is collected, it can be placed in a collection tubethat can include, among other things, a stabilizer (e.g.,ethylenediaminetetraacetic acid (EDTA)), an anticoagulant (e.g.,heparin, citrate dextrose, oxalate), an antimicrobial agent, a proteaseinhibitor, a buffer, a low amount (i.e., less than 0.05% w/v) of afixative, and combinations of these agents. Typically, a sample may bestored in a refrigerator (e.g., about 4° C.) for up two weeks or frozen(e.g., about −20° C. or less) for up to 2 months before it degrades to apoint beyond which optimal results may not be obtained when conductinganalyses on the sample.

Fixation of Rare Circulating Cells

Methods for enriching or isolating rare circulating cells, as describedherein, include fixing one or more rare circulating cells in a sample.In some embodiments, fixing one or more rare circulating cells in asample can create a fixed sample (i.e., a sample that includes one ormore fixed rare circulating cells). Fixing preserves cell structure andcan increase cell rigidity. Fixing can be achieved by varioustechniques, including physical methods (e.g., heating, micro-waving andcryo-preservation (i.e., freezing)) or chemical methods (e.g., additionof an alcohol or an aldehyde). There are two primary mechanisms by whichchemical fixation occurs: denaturation and cross-link formation.Denaturation can be induced by dehydrants, such as alcohols or acetone.These reagents can remove and replace free water in cells and tissues,which can render water soluble proteins insoluble. Non-coagulant fixingagents (e.g., aldehydes) can chemically react with proteins and othercell and tissue components and can become bound to them by addition andformation of inter-molecular and intra-molecular cross-links. Inpreferred embodiments, non-coagulant fixing agents can be used to fixrare circulating cells. In some instances, a fixative can include analdehyde, for example, formaldehyde, glutaraldehyde, orparaformaldehyde.

In some embodiments, a fixative can be added to a sample. In someembodiments, a fixative can be added to a sample so that a concentrationof the fixative in the sample equals a desired amount. For example, aconcentration of fixative in a sample can be at least 0.05% w/v, atleast 0.10% w/v, at least 0.15% w/v, at least 0.20% w/v, or at least0.25% w/v. In some embodiments, a concentration of fixative in a samplecan be at most 0.40% w/v, at most 0.35% w/v, at most 0.30% w/v, at most0.25% w/v, or at most 0.20% w/v. In some embodiments, a concentration offixative in a sample can be 0.05-0.35% w/v, preferably, 0.1-0.3% w/v,more preferably, about 0.25% w/v.

In certain circumstances, a predetermined amount of a fixative can beadded to a sample. In some embodiments, at least 50 μL, at least 100 μL,at least 150 μL, at least 200 μL, at least 250 μL, at least 500 μL or atleast 1 mL of a fixative can be added to a sample. In some embodiments,at most 2 mL, at most 1.5 mL, at most 1mL, at most 500 μL, at most 250μL, or at most 100 μL of a fixative can be added to a sample. In someembodiments, 50-500 μL, preferably, 20-500 μL, of a fixative can beadded to a sample.

In some embodiments, fixing one or more rare circulating cells in asample can include incubating the sample with a fixative. In someembodiments, a sample can be incubated with a fixative for at least 30seconds, at least 1 minute, at least 2 minutes, at least 5 minutes, orat least 10 minutes. In some embodiments, a sample can be incubated witha fixative for at most 30 minutes, at most 25 minutes, at most 20minutes, at most 15 minutes, or at most 10 minutes. In some embodiments,a sample can be incubated with a fixative for 1-20 minutes, preferably,3-10 minutes.

In some embodiments, fixing one or more rare circulating cells in asample can include incubating the sample with a fixative at atemperature of at least 0° C., at least 10° C., at least 20° C., atleast 30° C., or at least 40° C. In some embodiments, fixing one or morerare at least circulating cells in a sample can include incubating thesample with a fixative at a temperature of at most 75° C., at most 60°C., at most 50° C., at most 40° C., at most 30° C., or at most 20° C. Insome embodiments, fixing one or more rare at least circulating cells ina sample can include incubating the sample with a fixative at atemperature of 0° C.-40° C., 20° C.-30° C., or about room temperature.

When fixing one or more rare circulating cells in a sample, an amount offixative, an incubation time and an incubation temperature can eachinfluence the degree of fixation. Generally, adding more fixative to asample, increasing an incubation time, and/or increasing an incubationtemperature will increase the amount of fixation. Therefore, an increasein an incubation time may be able to compensate for a decreased amountof fixative in a sample. Conversely, an increase in an amount offixative in a sample may be able to compensate for a decreasedincubation time.

For rare circulating cells in a sample, however, a high degree offixation may not be desirable, as it can result in over-crosslinking ofsample components, which can in turn clog filtration devices, forexample. Therefore, in some embodiments, balancing these factors can beimportant for successful enrichment or isolation of rare circulatingcells from a sample.

In some embodiments, fixing one or more rare circulating cells in asample can include incubating the sample with a fixative (e.g.,formaldehyde) at 20-30° C. for at most 10 minutes, where a concentrationof fixative in a sample can be 0.1-0.35% w/v. In some embodiments,fixing one or more rare circulating cells in a sample can includeincubating the sample with a fixative (e.g., formaldehyde) at 20-30° C.for at most 30 minutes, where a concentration of fixative in a samplecan be 0.05-0.30% w/v.

In some cases, a fixative is added with other agents, such as a salinesolution (e.g., phosphate buffered saline), to a sample. A salinesolution can be added to a sample prior to a fixative to dilute thesample. A saline solution can be a buffer. In some embodiments, the pHof a sample (with or without a fixative and/or a saline solution) can bebetween pH 4 and pH 9, preferably, between pH 7 and pH 8.

Fixation can also be influenced by storage of a sample. For example, ablood sample that has been stored under refrigeration for a longerperiod of time (e.g., more than 5 days, more than 1 week or more than 2weeks) in a storage solution, particularly one with a low concentration(i.e., less than 0.05%) of a fixative may be incubated with either alower fixative concentration (e.g., 0.05-0.30% w/v, 0.05-0.20% w/v or0.05-0.15% w/v) and/or for a shorter time (e.g., at most 15 minutes, atmost 10 minutes, at most 5 minutes, or at most 2 minutes).

In some embodiments, fixing one or more rare circulating cells in asample to create a fixed sample can include fixing 50% or more, 60% ormore, 70% or more, 80% or more, or 90% or more of rare circulating cellsin the sample. A level of fixation can be quantified using a number oftechniques known in the art. For example, microscopy methods have beenused in previous studies to evaluate the performance of fixationmethods, including light microscopy (St-Laurent, et al. (2006)),reflection contrast microscopy (Hoetelmans, et al. (2001)), fluorescencemicroscopy (Celie, et al. (2005)), Raman microscopy (Meade, et al.(2010)), electron microscopy (Hoetelmans, et al. (2001)), as well asatomic force microscopy (Moloney, et al. (2004)). Atomic forcemicroscopy (AFM) has been widely used in all fields of surface sciencesince its invention in 1986, including microbiological studies(Bolshakova, et al. (2004)). (St-Laurent, et al., “Comparison of cellfixation methods of induced sputum specimens: an immunocytochemicalanalysis,” J Immunol Methods, 308(1-2):36-42, Jan. 20, 2006; Hoetelmans,et al., “Effects of acetone, methanol, or paraformaldehyde on cellularstructure, visualized by reflection contrast microscopy and transmissionand scanning electron microscopy,” Appl Immunohistochem Mol Morphol,9(4):346-51, Dec. 2001; Celie, et al., “Effect of fixation protocols onin situ detection of L-selectin ligands,” J Immunol Methods,298(1-2):155-9, Mar. 2005; Bonnier, et al., “Imaging live cells grown ona three dimensional collagen matrix using Raman microspectroscopy,”Analyst, 135(12):3169-772010, December 2010; Moloney, et al., “Atomicforce microscopy analysis of enveloped and non-enveloped viral entryinto, and egress from, cultured cells,” Ultramicroscopy, 100(3-4):163-9,August 2004; Bolshakova, et al., “Microbial surfaces investigated usingatomic force microscopy,” Biotechnol Prog., 20(6):1615-22,November-December 2004, each of which is incorporated by reference inits entirety).

Enrichment and/or Isolation of Rare Circulating Cells

Once one or more rare circulating cells in a sample have been fixed, theone or more fixed rare circulating cells can be enriched or isolated.Preferably, enrichment or isolation can be achieved using a sizeexclusion technique, which can enrich or isolate rare circulating cellsindependent of the level of heterogeneity within these cells. Anexample, of a size exclusion technique that can be utilized isfiltration. In some embodiments, enriching or isolating one or more rarecirculating cell can be performed by directing a fixed sample, includingone or more fixed rare circulating cells, through one or more filtermembranes.

A filter membrane can be made of any material that is compatible withrare circulating cells and/or a fixed sample. For example, a filtermembrane should be made of a material that does not bind or otherwiseinteract with components of a fixed sample. Additionally, a filtermembrane should be made of a material that will not chemically degradein the presence of a component of a fixed sample. A filter membranematerial can, for example, be made from a fibrous material, such assynthetic fibers (e.g., polyester or polypropylene), semi-syntheticfibers, regenerated fibers, or inorganic fibers. Examples of suitablefilter membrane materials can include cellulose acetate, nylon,polyethersulfone, regenerated cellulose, silicon, glass, polydimethylsiloxane, polyurethane, polyimide, polycarbonate,polytetrafluorethylene, and a metal (e.g., palladium). A filter membranecan include wetting agents, which should also be compatible with rarecirculating cells and/or a fixed sample.

A filter membrane can include one or more pores. A pore size of a filtermembrane can be less than a diameter of a rare circulating cell that isbeing enriched or isolated. In some embodiments, a filter membrane(e.g., at least one of one or more filter membranes) can have a poresize that is less than 50%, less than 40%, less than 30% or less than25% of a diameter of a rare circulating cell that is being enriched orisolated. For example, a filter with an 8 micron pore size can be usedto enrich or isolate circulating cancer cells that have a diameter ofabout 30 microns.

In some embodiments, a filter membrane (e.g., at least one of one ormore filter membranes) can have a pore size of at least 5 microns, atleast 6 microns, at least 7 microns, at least 8 microns, at least 9microns, or at least 10 microns. In some embodiments, a filter membrane(e.g., at least one of one or more filter membranes) can have a poresize of at most 12 microns, at most 11 microns, at most 10 microns, atmost 9 microns, at most 8 microns, at most 7 microns, or at most 6microns. In some embodiments, a filter membrane (e.g., at least one ofone or more filter membranes) can have a pore size of between 5 and 10microns, preferably, between 7 and 9 microns, more preferably 8 microns.

In some embodiments, one or more filter membranes can include a capturehole (i.e., pocket). A capture hole can have a size of at least 20microns, at least 25 microns, at least 30 microns, at least 40 micronsor at least 50 microns. A capture hole can have a size of at most 75microns, at least 60 microns, at least 50 microns, or at least 40microns. A pore can be included at the base of a capture hole.

Filter membranes can include pores of different sizes. For example, insome embodiments, a first filter membrane can have a first pore size anda second filter membrane can have a second pore size. In someembodiments, a first filter membrane can include a first pore having afirst pore size and a second pore having a second pore size.Combinations of these embodiments can be utilized.

One or more filter membranes can be part of a filtration device, forexample, a microfiltration device. In some instances, one or more filtermembranes can be part of a filter unit of a filtration device. Amicrofiltration device can also include, for example, a reservoir, acontrol unit and/or a waste outlet. A control unit can control the flowof a sample through a filtration device. A control unit can include, forexample, a valve, a pump, or a constrictor. In some cases, a controlunit can be an influx control unit. In some cases, a control unit can bean efflux control unit.

Isolating one or more rare circulating cell can be performed bydirecting a fixed sample, including one or more fixed rare circulatingcells, through one or more filter membranes. Directing a fixed samplethrough one or more filter membranes can result in retention of at least60%, at least 70%, at least 80%, at least 90%, or at least 95% of rarecirculating cells in the sample on the one or more filter membranes.

A variety of analyses can be performed on enriched or isolated rarecirculating cells. Examples of analyses include staining, dyeing,immunocytochemistry, immunohistochemistry, in situ hybridization, PCR,single cell sequence analysis, cell sorting, cell counting, microscopyor combination of these techniques.

EXAMPLES Example 1: Methods

A pretreatment method, including fixation, for filtration of rarecirculating cells from a blood sample was developed. In an exemplarymethod, blood was collected from a subject and put in a TransFix® bloodcollection for transport and storage. Prior to filtration, the collectedblood sample was transferred into a conical tube. The original bloodcollection tube was rinsed with a saline buffer to collect any residualcells, and the saline buffer was poured into the same conical tube asthe collected blood sample. After bringing up the total volume to 20 mLwith saline buffer, 250 μL of 16% formaldehyde was added to the dilutedblood sample to achieve a final formaldehyde concentration ofapproximately 0.2% w/v. The conical tube was pulse vortexed for 3seconds and incubated at room temperature for 5 minutes to fix thecells. The conical tube with the fixed blood sample was then loaded ontoan Integrated Cell Capture Micro-Device (ICCMD) filtration systemincluding an eight (8) micron filter. Isolated circulating tumor cellswere collected for further processing.

Example 2: Determination of Percent Cell Recovery

Blood samples were collected from subjects, as described in Example 1.The blood samples were then spiked with various known amounts ofNCI-H226 (a lung cancer cell line) cells and pre-treated according tothe method provided in Example 1. The fixed, spiked samples were loadedonto an ICCMD filtration system. NCI-H226 cells retained on the filterwere stained and counted. As shown in FIG. 1, there was a linearcorrelation between the number of NCI-H226 cells spiked into each bloodsample and the number of NCI-H226 cells recovered from each bloodsample. Comparison of the number of NCI-H226 cells spiked into eachblood sample to the number of NCI-H226 cells recovered from each bloodsample demonstrates that approximately 80% of recovery was achieved whenthe pretreatment method was utilized. The data represented animprovement over prior methodologies that do not include a fixativepretreatment, which generally have recovery rates of about 40-60% forspiked NCI-H226 cells (see, e.g., bar representing 0% w/v fixative inFIG. 2).

Example 3: Optimization of Percent Fixative

Various concentrations of fixative were tested to determine optimalconcentrations of fixative for achieving an increased recovery of rarecirculating cells (e.g., circulating tumor cells) from blood samples.Blood samples were collected from subjects, as described in Example 1.Each of the blood samples was spiked with the same known amount ofNCI-H226 cells. The spiked blood samples were then pretreated asdescribed in Example 1 using different concentrations of formaldehyde.The samples were loaded onto an ICCMD filtration system, and NCI-H226cells retained on the filter were stained and counted. As shown in FIG.2, the control sample, to which fixative was not added, showed arecovery rate of approximately 52%. Adding fixative improved therecovery rate, with the sample including 0.25% w/v formaldehyde showingthe highest recovery rate (˜87%). While two other higher concentrations,0.3% w/v and 0.35% w/v, showed improved recovery rates over the controlsample, use of 0.3% w/v and 0.35% w/v resulted in failure about half thetime, possibly due to clogging of the filtration system. Without beingbound to any theory, a possible reason for improved recovery of cancercells is that an optimized fixation increases the rigidity of the cancercells, which in turn increases retention of those cells above the poresof the membrane.

EQUIVALENTS

It is to be understood that while the disclosure has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

All patents, applications, and publications cited in the text above areincorporated herein by reference.

What is claimed is:
 1. A method for isolating rare circulating cellsfrom a blood sample of a subject, comprising: fixing one or more rarecirculating cells in a blood sample to create a fixed blood sample; andisolating the one or more rare circulating cells by directing the fixedblood sample through one or more filter membranes.
 2. The method ofclaim 1, wherein the rare circulating cells are circulating tumor cells.3. The method of claim 2, wherein the circulating tumor cells originatedfrom a carcinoma, sarcoma, leukemia, lymphoma, myeloma or nervous systemcancer.
 4. The method of claim 3, wherein the circulating tumor cellsoriginated from a carcinoma.
 5. The method of any one of the precedingclaims, wherein the subject is a mammal.
 6. The method of any one of thepreceding claims, wherein the subject is a human.
 7. The method of anyone of the preceding claims, wherein fixing the one or more rarecirculating cells in the blood sample comprises adding a fixative to theblood sample.
 8. The method of claim 7, wherein the fixative includes analdehyde.
 9. The method of claim 7 or 8, wherein the fixative includesformaldehyde, glutaraldehyde, or paraformaldehyde.
 10. The method of anyone of claims 7-9, wherein the fixative includes formaldehyde.
 11. Themethod of any one of claims 7-10, wherein fixing the one or more rarecirculating cells in the blood sample comprises adding a fixative to theblood sample so that the concentration of the fixative in the bloodsample is 0.05-0.35% w/v.
 12. The method of any one of claims 7-11,wherein fixing the one or more rare circulating cells in the bloodsample comprises adding a fixative to the blood sample so that theconcentration of the fixative in the blood sample is 0.1-0.3% w/v. 13.The method of any one of claims 7-12, wherein fixing the one or morerare circulating cells in the blood sample comprises adding a fixativeto the blood sample so that the concentration of the fixative in theblood sample is about 0.25% w/v.
 14. The method of any one of claims7-13, wherein fixing the one or more rare circulating cells in the bloodsample comprises incubating the blood sample with the fixative for 1-20minutes.
 15. The method of any one of claims 7-14, wherein fixing theone or more rare circulating cells in the blood sample comprisesincubating the blood sample with the fixative for 3-10 minutes.
 16. Themethod of any one of the preceding claims, wherein the one or morefilter membranes are part of a microfiltration device.
 17. The method ofany one of the preceding claims, wherein at least one of the one or morefilter membranes has a pore size between 5 and 10 microns.
 18. A methodof enriching rare circulating cells in a blood sample of a subject,comprising: fixing one or more circulating cells in the blood sample tocreate a fixed blood sample; and enriching the one or more rarecirculating cells by directing the fixed blood sample through one ormore filter membranes.
 19. A method of analyzing rare circulating cellsin a blood sample of a subject, comprising: fixing one or more rarecirculating cells in a blood sample to create a fixed blood sample;enriching or isolating one or more rare circulating cells by directingthe fixed blood sample through one or more filter membranes; andperforming an analysis of the one or more rare circulating cellsincluding staining, dyeing, immunocytochemistry, immunohistochemistry,in situ hybridization, PCR, single cell sequence analysis, cell sorting,cell counting, microscopy or a combination thereof.
 20. A compositioncomprising: one or more rare circulating cells from a subject; and afixative, wherein the concentration of fixative in the composition is0.05-0.35% w/v.
 21. The composition of claim 20, wherein the one or morecirculating cells are from the blood of the subject.